Purification and characterization of protease from intestine of tilapia

Tilapia intestines as enzyme raw materials are studied systematically by ultrasonic extraction technology of auxiliary, electrophoresis, chromatography techniques and so on for the first time. The results showed that: after organic mashing and ultrasound-assisted extracting, 30％－70% of ammonium sulfate fractions, QFF anion chromatography and Sephedax G-100 gel chromatography, the purified protease from tilapia intestines was obtained. The purified protease specific activity is 335 U/mg and yield is 32.8%. The result of SDS-PAGE showed a single band and the subunit molecular weight of tilapia intestines protease was 28 ku. The protease optimum pH value and temperature are 8.0－8.5 and 37－42 ℃ respectively. It is stable at pH range from 7.0 to 9.0 and has good thermal stability. Enzyme K_m value and V_maxvalue are 0.605 g/L and 9.407 μg/min. Ag⁺ and Pb²⁺ can inhibit the enzymatic activity completely while Na⁺ and K⁺ without inhibition. PMSF can strongly inhibit the activity of the enzyme. Pepstatin A and urea partly inhibit the activity of the enzyme. EDTA has no effect on the enzyme while DTT can activate the enzyme activity. The protease from tilapia intestines is a kind of serine protease. Further work should be carried out on the sequences of the enzyme and its applications．